High-resolution scanning microscopy

ABSTRACT

A microscope and method for high resolution scanning microscopy of a sample, having an illumination device, an imaging device for the purpose of scanning at least one point or linear spot across the sample and of imaging the point or linear spot into a diffraction-limited, static single image below a reproduction scale in a detection plane. A detector device is used for detecting the single image in the detection plane for various scan positions, with a location accuracy which, taking into account the reproduction scale in at least one dimension/measurement, is at least twice as high as a full width at half maximum of the diffraction-limited single image.

RELATED APPLICATIONS

The present application is a continuation of application Ser. No.14/457,833 filed on Aug. 12, 2014, which is a non-provisional ofProvisional Application No. 62/025,649 filed on Jul. 17, 2014 and claimspriority benefit of German Application No. DE 10 2013 013 792.6 filed onAug. 15, 2013 and German Application No. DE 10 2013 019 347.8 filed onNov. 15, 2013, the contents of each are incorporated by reference intheir entirety.

FIELD OF THE INVENTION

The invention relates to a microscope for high resolution scanningmicroscopy of a sample, having an illumination device for the purpose ofilluminating the sample, an imaging device for the purpose of scanning apoint or linear spot across the sample and of imaging the point- orlinear spot into a diffraction-limited, resting single image, with areproduction scale in a detection plane, a detector device for thepurpose of detecting the single image in the detection plane for variousscan positions, with a location accuracy which, taking into account thereproduction scale, is at least twice as high as a full width at halfmaximum of the diffraction-limited single image, an evaluation devicefor the purpose of evaluating a diffraction structure of the singleimage for the scan positions, using data from the detector device, andfor the purpose of generating an image of the sample which has aresolution which is enhanced beyond the diffraction limit. The inventionfurther relates to a method for high resolution scanning microscopy of asample, wherein the sample is illuminated, wherein a point- or linearspot guided over the sample in a scanning manner is imaged into a singleimage, wherein the spot is imaged into the single image, with areproduction scale, and diffraction-limited, and the single image isstatic in a detection plane, wherein the single image is detected forvarious different scan positions with a location accuracy which is atleast twice as high, taking into account the reproduction scale, as afull width at half maximum of the diffraction-limited single image, suchthat a diffraction structure of the single image is detected, whereinfor each scan position, the diffraction structure of the single image isevaluated and an image of the sample is generated which has a resolutionwhich is enhanced beyond the diffraction limit.

BACKGROUND OF THE INVENTION

Such a microscope and/or microscopy method is known, by way of example,from the publication C Mueller and J. Enderlein, Physical ReviewLetters, 104, 198101 (2010), or EP 2317362 A1, which also lists furtheraspects of the prior art.

This approach achieves an increase in location accuracy by imaging adiffraction-limited spot on a detection plane. The diffraction-limitedimaging process images a point spot as an Airy disk. This diffractionspot is detected in the detection plane in such a manner that itsstructure can be resolved. Consequently, an oversampling is realized atthe detector with respect to the imaging power of the microscope. Theshape of the Airy disk is resolved in the imaging of a point spot. Witha suitable evaluation of the diffraction structure—which is detailed inthe documents named, the disclosure of which in this regard is herebycited in its entirety in this application—an increase in resolution by afactor of 2 beyond the diffraction limit is achieved.

However, it is unavoidable in this case, for the detector, that it isnecessary to capture a single image with multiple times more imageinformation for each point on the sample which is scanned in this way,compared to a conventional laser scanning microscope (shortened to ‘LSM’below). If the structure of the single image of the spot is detected, byway of example, with 16 pixels, not only is the volume of data per spot16-times higher, but also a single pixel also contains, on average, only1/16 of the radiation intensity which would fall on the detector of anLSM in a conventional pinhole detection. Because the radiation intensityis of course not evenly distributed across the structure of the singleimage—for example the Airy disk—in reality even less, and particularlysignificantly less—radiation intensity arrives at the edge of thisstructure than the average value of 1/n for n pixels.

Consequently, the problem exists of being able to detect quantities ofradiation at the detector at high resolution. ConventionalCharge-Coupled Device (CCD) arrays which are typically used inmicroscopy do not achieve sufficient signal-to-noise ratios, such thateven a prolongation of the duration for the image capture, which wouldalready be disadvantageous in application per se, would not providefurther assistance. Avalanche Photodetector (APD) arrays also sufferfrom excessively high read noise, such that a prolongation of themeasurement duration here as well would result in an insufficientsignal/noise ratio. The same is true for complementary metal-oxidesemiconductor (CMOS detectors), which also are disadvantageous withregards to the size of the detector element, because thediffraction-limited single image of the spot would fall on too fewpixels. Photomultiplier Tube (PMT) arrays suffer from similarconstructed space problems. The pixels in this case are likewise toolarge. The constructed space problems are particularly a result of thefact that an implementation of a microscope for high resolution can onlybe realized, as far as the effort required for development and thedistribution of the device, are concerned, if it is possible tointegrate the same into existing LSM constructions. However, specificsizes of the single images are prespecified in this case. As a result, adetector with a larger surface area could only be installed if a lenswere additionally configured which would enlarge the image once more toa significant degree—that is, several orders of magnitude. Such a lensis very complicated to design in cases where one wishes to obtain thediffraction-limited structure without further imaging errors.

Other methods are known in the prior art for high resolution, whichavoid the problems listed above which occur during detection. By way ofexample, a method is mentioned in EP 1157297 B1, wherein non-linearprocesses are exploited using structured illumination. A structuredillumination is positioned over the sample in multiple rotary and pointpositions, and the sample is imaged on a wide-field detector in thesedifferent states in which the limitations listed above do not exist.

A method which also achieves high resolution without the detectorlimitations listed above (that is, a resolution of a sample image beyondthe diffraction limit) is known from WO 2006127692 and DE 102006021317.This method, abbreviated as ‘PALM’, uses a marking substance which canbe activated by means of an optical excitation signal. Only in theactivated state can the marking substance be stimulated to releasecertain fluorescence radiation by means of excitation light. Moleculeswhich are not activated do not emit fluorescent radiation, even afterillumination with excitation light. The excitation light thereforeswitches the marking substance into a state in which it can bestimulated to fluoresce. Therefore, this is generally termed a‘switching signal’. The same is then applied in such a manner that atleast a certain fraction of the activated marking molecules are spacedapart from neighboring, likewise-activated marking molecules in such amanner that the activated marking molecules are separated on the scaleof the optical resolution of the microscope, or can be separatedretroactively. This is termed ‘isolation’ of the activated molecules. Itis easy, for these isolated molecules, to determine the center of theirradiation distribution which is limited by the resolution, and totherefore determine the location of the molecules using calculation,with a higher precision than the optical imaging actually allows. Toimage the entire sample, the PALM method takes advantage of the factthat the probability of a marking molecule being activated by theswitching signal at a given intensity of the switching signal is thesame for all of the marking molecules. The intensity of the switchingsignal is therefore applied in such a manner that the desired isolationresults. This method step is repeated until the greatest possible numberof marking molecules have been excited [at least] one time within afraction which has been excited to fluorescence.

SUMMARY OF THE INVENTION

In the invention, the spot sampled on the sample is imaged statically ina detection plane. The radiation from the detection plane is thenredistributed in a non-imaging manner and directed to the detectorarray. The term “non-imaging” in this case refers to the single imagepresent in the detection plane. Individual regions of the area of thissingle image can of course, however, be imaged within the laws ofoptics. As such, imaging lenses can naturally be placed between thedetector array and the redistribution element. The single image in thedetection plane, however, is not preserved as such in theredistribution.

The term ‘diffraction-limited’ should not be restricted here to thediffraction limit according to Abbe's Theory. Rather, it should alsoencompass situations in which the configuration fails to reachtheoretical maximum by an error of 20%, due to actual insufficiencies orlimitations. In this case as well, the single image has a structurewhich is termed a “diffraction structure” in this context. It isoversampled.

This principle makes it possible to use a detector array, the size ofwhich does not match the single image. The detector array isadvantageously larger or smaller in one dimension that the single imagebeing detected. The idea of the different geometric configurationincludes both a different elongation of the detector array and anarrangement with a different aspect ratio with respect to the height andwidth of the elongation of the single image in the detection plane. Thepixels of the detector array can additionally be too large for therequired resolution. It is also allowable, at this point, for theoutline of the pixel arrangement of the detector array to befundamentally different than the outline which the single image has inthe detection plane. In any case, the detector array according to theinvention has a different size than the single image in the detectionplane. The redistribution in the method and/or the redistributionelement in the microscope make it possible to select a detector arraywithout needing to take into account the dimensional limitations andpixel size limitations which arise as a result of the single image andits size. In particular, it is possible to use a detector row as adetector array.

The image of the sample is created from a plurality of single images, inthe conventional LSM manner by scanning the sample with the spot,wherein each of the single images is associated with another sampleposition—that is, another scan position.

The concept of the invention can also be carried out at the same timefor multiple spots in a parallelized manner, as is known for laserscanning microscopy. In this case, multiple spots are sampled on thesample in a scanning manner, and the single images of the multiple spotslie next to each other statically in the detection plane. They are theneither redistributed by a shared redistribution element which isaccordingly large with respect to surface area, and/or by multipleindividual redistribution elements, then relayed to an accordingly largesingle detector array and/or to multiple individual detector arrays.

The subsequent description focuses, by way of example, on the samplingprocess using an individual spot. However, this should not be understoodas a limitation, and the described features and principles apply in thesame manner for the parallel sampling of multiple point spots, as wellas to the use of a linear spot. The latter case is of course onlydiffraction-limited in the direction perpendicular to the elongation ofthe line, such that the features of this description with regards tothis aspect only apply in one direction (perpendicular to the elongationof the line).

With the procedure according to the invention, the LSM method can becarried out at a satisfactory speed and with acceptable complexity ofthe apparatus. The invention opens up a wide field of applications for ahigh resolution microscopy principle which has not existed to date.

One possibility for realizing the redistribution and/or theredistribution element consists of using a bundle of optical fibers.These can preferably be designed as multi-mode optical fibers. Thebundle has an input which is arranged in the detection plane and whichhas an adequate dimensioning for the dimensions of thediffraction-limited single image in the detection plane. In contrast, atthe output, the optical fibers are arranged in the geometric arrangementwhich is prespecified by the detector array, and which differs from theinput. The output ends of the optical fibers in this case can be guideddirectly to the pixels of the detector array. It is particularlyadvantageous if the output of the bundle is gathered in a plug which canbe easily plugged into a detector row—for example an APD or PMT row.

It is important for the understanding of the invention to differentiatebetween pixels of the detector array and the image pixels with which thesingle image is resolved in the detection plane. Each image pixel isgenerally precisely functionally assigned to one pixel of the detectorarray. However, the two are different with respect to their arrangement.Among other things, it is a characterizing feature of the inventionthat, in the detection plane, the radiation is captured on image pixelswhich produce an oversampling of the single image with respect to theirsize and arrangement. In this manner, the structure of the single imageis resolved which, due to the diffraction-limited production of thesingle image, is a diffraction structure. The redistribution element hasan input side on which this image pixel is provided. The input side liesin the detection plane. The redistribution element directs the radiationon each image pixel to one of the pixels of the detector array. Theassignment of image pixels to pixels of the detector array does notpreserve the image structure, which is why the redistribution isnon-imaging with respect to the single image. The invention couldtherefore also be characterized in that, in a microscope of the class,the detector device has a non-imaging redistribution element which hasinput sides in the detection plane, at which the radiation is capturedby means of image pixels. The redistribution element further has anoutput side on which the radiation captured at the image pixels isrelayed to pixels of a detector array, wherein the radiation isredistributed from the input side to the output side in a non-imagingmanner with respect to the single image. In an analogous manner, themethod according to the invention could be characterized in that, in amethod of the class, the radiation is captured in the detection plane bymeans of image pixels which are redistributed to pixels of the detectorarray in a non-imaging manner with respect to the single image. Thedetector array differs from the arrangement and/or the size of the imagepixels in the detection plane as regards the arrangement and/or size ofits pixels. In addition, the image pixels in the detection plane areprovided by the redistribution element in such a manner that, withrespect to the diffraction limit, the diffraction structure of thesingle image is oversampled.

In highly-sensitive detector arrays, it is known that adjacent pixelsdemonstrate interference when radiation intensities are high, as aresult of crosstalk. To prevent this, an implementation is preferred inwhich the optical fibers are guided from the input to the output in sucha manner that optical fibers which are adjacent at the output are alsoadjacent at the input. Because the diffraction-limited single image doesnot demonstrate any large jumps in radiation intensity changes, such aconfiguration of the redistribution element automatically ensures thatadjacent pixels of the detector array receive the least possibledifferences in radiation intensity, which minimizes crosstalk.

In place of a redistribution based on optical fibers, it is alsopossible to equip the redistribution element with a mirror which hasmirror elements with different inclinations. Such a mirror can bedesigned, by way of example, as a multi-faceted mirror, a DigitalMicromirror Device (DMD), or adaptive mirror, wherein in the latter twovariants, a corresponding adjustment and/or control process ensures theinclination of the mirror elements. The mirror elements direct theradiation from the detection plane to the pixels of the detector array,the geometrical design of which is different from the mirror elements.

The mirror elements depict—as do the optical fiber ends at the input ofthe optical fiber bundle—the image pixels with respect to the resolutionof the diffraction structure of the single image. Their size is decisivefor the oversampling. The pixel size of the detector array is not. As aresult, a group of multiple single detectors is understood in this caseto be a detector array, because they always have a different arrangement(that is—a larger arrangement) than the image pixels in the detectionplane.

In LSM, different lenses are used depending on the desired resolution.An exchange of a lens changes the dimensions of a single image in thedetection plane. For this reason, it is preferred that a zoom lens isarranged in front of the detection plane in the direction of imaging forthe purpose of matching the size of the single image to the size of thedetector device. Such a zoom lens varies the size of the single image ina percent range which is significantly smaller than 100%, and istherefore much simpler to implement than a multiplication of the size ofthe single image—which was described as disadvantageous above.

The illumination of the sample is preferably carried out as in a typicalLSM process, likewise scanning—although this is not absolutelynecessary. However, the maximum increase in resolution is achieved inthis way. If the sample is illuminated in a scanning manner, it isadvantageous that the illumination device and the imaging device have ashared scanning device which guides an illumination spot across thesample, and simultaneously descans the spot at which the sample isimaged, which is coincident with the illumination spot, with respect tothe detector, such that the single image is static in the detectionplane. In such a construction, the zoom lens can be placed in the sharedpart of the illumination device and imaging device. The lens then makesit possible to not only match the single image to the size of thedetector in the detection plane, but also it additionally enables theavailable illumination radiation to be coupled into the lens pupilcompletely, without edge loss, wherein said lens pupil can vary togetherwith the selection of the lens.

A radiation intensity-dependent crosstalk between adjacent pixels of thedetector array can, as already explained, be reduced during theredistribution by means of an optical fiber bundle by a suitablearrangement of the optical fibers in the bundle. In addition oralternatively thereto, it is also possible to carry out a calibration.For this purpose, each optical fiber receives radiation one after theother, and the interference signal is detected in neighboring pixels. Inthis manner, a calibration matrix is established, by means of which aradiation intensity-dependent crosstalk between adjacent pixels iscorrected in the later microscopy of the sample.

The resolution of the diffraction structure of the single image alsomakes it possible to determine a direction of movement of the spot,wherein the sample is moved along the same during the scanning. Thisdirection of movement is known in principle from the mechanism of thescanner (for example, a scanning mirror or a moving sample table), butnevertheless there are residual inaccuracies in this case arising fromthe mechanism. These can be eliminated by evaluating signals ofindividual pixels of the detector array by means of cross-correlation.In this case, one takes advantage of the fact that, related adjacentimage pixels in the sample overlap to a certain degree due to thediffraction-limited imaging of the spot, whereas their centers lieadjacent to each other. If the signals of such image pixels aresubjected to a cross-correlation, it is possible to reduce and/or tocompletely eliminate a residual inaccuracy which persists as a result ofunavoidable tolerances of the scanning mechanism.

In addition to the increased resolution, it is possible to detect achronological change in the fluorescence in the detection volumecomprised by the spot via the spatial and chronological correlation ofthe signals from a series of measurements of the individual detectorelements (to which the image pixels in the detection plane arefunctionally assigned). By way of example, diffusion coefficients can bedetermined from a chronological correlation, as in fluorescencecorrelation spectroscopy, and oriented diffusion and diffusion barrierscan be visualized by incorporating the spatial correlation between imagepixels. Movement processes of the fluorescence molecules are also ofgreat interest for tracking applications as well, because theillumination spot in this case should follow the movement of thefluorescent molecules. The arrangement described here makes it possibleto determine the movement direction with high precision, even during thebleaching time of a pixel. For this reason, it is preferred, as oneimplementation, that changes in the sample are detected by means ofdetermining and evaluating a chronological change in thediffraction-limited single image for the point- or linear spot which isstationary in the sample.

The procedure according to the invention also makes it possible tomodify the illumination distribution in scanning illuminationprocesses—for example by means of a phase filter. The method asdescribed in Gong et al., Opt. Let., 34, 3508 (2009) can be realizedvery easily as a result.

Where a method is described herein, a control device implements thismethod in the operation of the microscope.

It should be understood that the features named above and explainedfurther below can be used not only in the given combinations, but alsoin other combinations or alone without departing from the scope of thepresent invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is described in greater detail below with reference to theattached drawings, which also disclose essential features of theinvention, wherein:

FIG. 1 shows a schematic illustration of a laser scanning microscope forhigh resolution microscopy,

FIG. 2 shows an enlarged illustration of a detector device of themicroscope in FIG. 1,

FIG. 3 and FIG. 4 show top views of possible embodiments of the detectordevice 19 in a detection plane,

FIG. 5 shows one implementation of the microscope in FIG. 1 using a zoomlens, for the purpose of adapting the size of the detector field,

FIG. 6 shows a modification of the microscope in FIG. 5 with respect tothe zoom lens and with respect to a further implementation formulti-color imaging,

FIG. 7 shows a modification of the microscope in FIG. 1, wherein themodification pertains to the detector device,

FIG. 8 shows a modification of the detector device 19 in FIG. 7,

FIG. 9 shows a schematic illustration of a laser scanning microscope asdescribed above with reference to the illustrations in FIGS. 1-8, withsub-Airy resolution detection in the pinhole plane, and a multi-fiberbundle arrangement,

FIG. 10 illustrates a further embodiment with slider,

FIG. 11 shows a modification of the microscope of FIG. 10,

FIG. 12A shows sampling of the detection light and binning with 128fibres,

FIG. 12B shows sampling of the detection light and binning with one Airyimaged on 128 fibres,

FIG. 12C shows sampling of the detection light and binning with twoAirys on the 128 fibres, and

FIG. 12D shows sampling of the detection light and binning with 1.5Airys on the 128 fibres.

DESCRIPTION OF THE EMBODIMENTS

FIG. 1 schematically shows a laser scanning microscope 1 which isdesigned for the purpose of microscopy of a sample 2. The laser scanningmicroscope (abbreviated below as LSM) 1 is controlled by a controldevice C and comprises an illumination beam path 3 and an imaging beampath 4. The illumination beam path illuminates a spot in the sample 2,and the imaging beam path 4 images this spot, subject to the diffractionlimit, for the purpose of detection. The illumination beam path 3 andthe imaging beam path 4 share a plurality of elements. However, this islikewise less necessary than a scanned spot illumination of the sample2. The same could also be illuminated in wide-field.

The illumination of the sample 2 in the LSM 1 is carried out by means ofa laser beam 5 which is coupled into a mirror 8 via a deflection mirror6, which is not specifically functionally necessary, and a lens 7. Themirror 8 functions so that the laser beam 5 falls on an emission filter9 at a reflection angle. To simplify the illustration, only the primaryaxis of the laser beam 5 is drawn for the same.

Following the reflection on the emission filter 9, the laser beam 5 isdeflected biaxially by a scanner 10, and focused by means of lenses 11and 12 through an objective 13 to a spot 14 in the sample 2. The spot inthis case is point-shaped in the illustration in FIG. 1, but a linearspot is also possible. Fluorescence radiation excited in the spot 14 isrouted via the objective 13, the lenses 11 and 12, and back to thescanner 10, after which a static light beam once more is present in theimaging direction. This passes through the emission filters 9 and 15,which have the function of selecting the fluorescence radiation in thespot 14, with respect to the wavelength thereof, and particularly ofseparating the same from the illumination radiation of the laser beam 5,which can serve as excitation radiation, by way of example. A lens 16functions so that the spot 14 overall is imaged into adiffraction-limited image 17 which lies in a detection plane 18. Thedetection plane 18 is a plane which is conjugated to the plane in whichthe spot 14 in the sample 2 lies. The image 17 of the spot 14 iscaptured in the detection plane 18 by a detector device 19 which isexplained in greater detail below in the context of FIGS. 2 to 4. Inthis case, it is essential that the detector device 19 spatiallyresolves the diffraction-limited image 17 of the spot 14 in thedetection plane 18.

The intensity distribution of the spot over the detection cross-section(the Gaussian distribution) in 18 is illustrated below as 18 a in FIG.1.

The control device C controls all components of the LSM 1, particularlythe scanner 10 and the detector device 19. The control device capturesthe data of each individual image 17 for different scan positions,analyzes the diffraction structure thereof, and generates a highresolution composite image of the sample 2.

The LSM 1 in FIG. 1 is illustrated by way of example for a single spotwhich is scanned on the sample. However, it can also be used for thepurpose of scanning according to a linear spot which extends, by way ofexample, perpendicular to the plane of the drawing in FIG. 1. It is alsopossible to design the LSM 1 in FIG. 1 in such a manner that multipleadjacent point spots in the sample are scanned. As a result, theircorresponding single images 17 lie in the detection plane 18, likewiseadjacent to each other. The detector device 19 is then accordinglydesigned to detect the adjacent single images 17 in the detection plane18. An extended plug 23, detector array 24 and pixels 25 are describedin more detail below in relation to FIG. 2.

The detector device 19 is illustrated in an enlarged fashion in FIG. 2.It consists of an optical fiber bundle 20 which feeds a detector array24. The optical fiber bundle 20 is built up of individual optical fibers21. The ends of the optical fibers 21 form the optical fiber bundleinput 22, which lies in the detection plane 18. The individual ends ofthe optical fibers 21 therefore constitute pixels by means of which thediffraction-limited image 17 of the spot 14 is captured. Because thespot 14 in the embodiment in FIG. 1 is, by way of example, a point spot,the image 17 is an Airy disk, the size of which remains inside thecircle which, in FIGS. 1 and 2, represents the detection plane 18. Thesize of the optical fiber bundle input 22 is therefore such, that thesize of the Airy disk is covered thereby. The individual optical fibers21 in the optical fiber bundle 20 are given a different geometricarrangement at their outputs than at the optical fiber bundle input 22,particularly in the form of an extended plug 23 in which the output endsof the optical fibers 21 lie adjacent to each other. The plug 23 isdesigned to match the geometric arrangement of the detector array 24(i.e., the detector row)—that is, each output end of an optical fiber 21lies precisely in front of a pixel 25 of the detector array 24.

The geometric dimensions of the redistribution element are entirelyfundamental—meaning that they are matched on the input side thereof tothe dimensions of the single image (and/or, in the case of multiplepoint-spots, to the adjacent single images, regardless of theimplementation of the redistribution element, which is made in FIG. 4 byan optical fiber bundle). The redistribution element has the function ofcapturing the radiation from the detection plane 18, in such a mannerthat the intensity distribution of the single image 17, measured by thesampling theorem, is oversampled with respect to the diffraction limit.The redistribution element therefore has pixels (formed by the inputends of the optical fibers in the construction shown in FIG. 3) lying inthe detection plane 18, which are smaller by at least a factor of 2 thanthe smallest resolvable structure which is produced in the detectionplane 18 from the diffraction limit, taking into account thereproduction scale.

Of course, the use of a plug 23 is only one of many possibilities forarranging the output ends of the optical fibers 21 in front of thepixels 25. It is equally possible to use other connections. In addition,the individual pixels 25 can be directly fused to the optical fibers 21.It is not at all necessary to use a detector row 24. Rather, anindividual detector can be used for each pixel 25.

FIGS. 3 and 4 show possible embodiments of the optical fiber bundleinput 22. The optical fibers 21 can be melted together at the opticalfiber bundle input 22. In this way, a higher fullness factor isachieved—meaning that holes between the individual optical fibers 21 atthe optical fiber bundle input 22 are minimized. The melting would alsolead to a certain crosstalk between adjacent optical fibers. If onewould like to prevent this, the optical fibers can be glued. Arectangular arrangement of the ends of the optical fibers 21 is alsopossible, as FIG. 4 shows.

The individual optical fibers 21 are preferably functionally assigned tothe individual pixels 25 of the detector array 24 in such a manner thatoptical fibers 21 positioned adjacent to each other at the optical fiberbundle input 22 are also adjacent at the detector array 24. By means ofthis approach, crosstalk in minimized between adjacent pixels 25,wherein said crosstalk can arise, by way of example, from scatterradiation or during the signal processing of the individual pixels 25.If the detector array 24 is a row, the corresponding arrangement can beachieved by fixing the sequence of the individual optical fibers on thedetector row using a spiral which connects the individual optical fibersone after the other in the perspective of a top view of the detectionplane 18.

FIG. 3 further shows blind fibers 26 which lie in the corners of thearrangement of the optical fibers 21 at the optical fiber bundle input22. These blind fibers are not routed to pixels 25 of the detectorarray. At the positions of the blind fibers, there would no longer beany signal intensity required for the evaluation of the signals. As aresult, one can reduce the number of the optical fibers 21, andtherefore the number of the pixels 25 in the detector row 24 or thedetector array, in such a manner that it can be possible to work with 32pixels, by way of example. Such detector rows 24 are already used inother ways in laser scanning microscopy, with the advantage that onlyone signal evaluation electronic unit needs to be installed in suchlaser scanning microscopes, and a switch is then made between anexisting detector row 24 and the further detector row 24 which is addedby the detector device 19.

According to FIG. 4, optical fibers with a square base shape are usedfor the bundle. They likewise have a high degree of coverage in thedetection plane, and therefore efficiently collect the radiation.

FIG. 5 shows one implementation of the LSM 1 in FIG. 1, wherein a zoomlens 27 is arranged in front of the detection plane 18. The conjugatedplane in which the detection plane 18 was arranged in the constructionshown in FIG. 1 now forms an intermediate plane 28, from which the zoomlens 27 captures the radiation and relays the same to the detectionplane 18. The zoom lens 27 makes it possible for the image 17 to beoptimally matched to the dimensions of the input of the detector device19.

FIG. 6 shows yet another modification of the laser scanning microscope 1in FIG. 1. On the one hand, the zoom lens is arranged in this case asthe zoom lens 29, in such a manner that it lies in a part of the beampath, the same being the route of both the illumination beam path 3 andthe imaging beam path 4. As a result, the advantage is accrued that notonly the size of the image 17 on the input side of the detector device19 can be adapted, but also that the pupil fullness of the lens 13,relative to the imaging beam path 4, and therefore the exploitation ofthe laser beam 5, can be adapted as well.

In addition, the LSM 1 in FIG. 6 also has a two-channel design, as aresult of the fact that a beam splitter is arranged downstream of theemission filter 9, and separates the radiation into two separate colorchannels. The corresponding elements of the color channels eachcorrespond to the elements which are arranged downstream of the emissionfilter 9 in the imaging direction in the LSM 1 in FIG. 1. The colorchannels are differentiated in the illustration in FIG. 6 by thereference number suffixes “a” and/or “b”.

Of course, the implementation using two color channels is independent ofthe use of the zoom lens 29. However, the combination has the advantagethat a zoom lens 27 which would need to be independently included ineach of the color channels, and therefore would be present twice, isonly necessary once. Of course, the zoom lens 27 can also, however, beused in the construction according to FIG. 1, and the LSM 1 in FIG. 6can also be realized without the zoom lens 29.

FIG. 7 shows a modification of the LSM 1 in FIG. 1, with respect to thedetector device 19.

The detector device 19 now has a multi-facet mirror 30 which carriesindividual facets 31. The facets 31 correspond to the ends of theoptical fibers 21 at the optical fiber bundle input 22 with respect tothe resolution of the image 17. The individual facets 31 differ withrespect to their inclination from the optical axis of the incident beam.Together with a lens 32 and a mini-lens array 33, as well as a deflectormirror 34 which only serves the purpose of beam folding, each facet 31reproduces a surface area segment of the single image 17 on one pixel 25of a detector array 24. Depending on the orientation of the facets 31,the detector array 24 in this case can preferably be a 2D array.However, a detector row is also possible.

FIG. 8 shows one implementation of the detector device 19 in FIG. 7,wherein a refractive element 35 is still arranged in front of the lens32, and distributes the radiation particularly well to a detector row.

The detector array 24 can, as already mentioned, be selected based onits geometry, with no further limitations. Of course, the redistributionelement in the detector device 19 must then be matched to thecorresponding detector array. The size of the individual pixels withwhich the image 17 is resolved is also no longer prespecified by thedetector array 24, but rather by the element which produces theredistribution of the radiation from the detection plane 18. For an Airydisk, the diameter of the disk in a diffraction-limited image is givenby the formula 1.22·λ/NA, wherein λ is the average wavelength of theimaged radiation, and NA is the numerical aperture of the lens 13. Thefull width at half maximum is then 0.15·λ/NA. In order to achieve highresolution, it is sufficient for location accuracy of the detection tobe made twice as high as the full width at half maximum—meaning that thefull width at half maximum is sampled twice. A facet element 31 and/oran end of an optical fiber 21 at the optical fiber bundle input 22 maytherefore be, at most, half as large as the full width at half maximumof the diffraction-limited single image. This of course is true takinginto account the reproduction scale which the optics behind the lens 13produces. In the simplest case, a 4×4 array of pixels in the detectionplane 18 per full width at half maximum would thereby be more thanadequate.

The zoom lens which was explained with reference to FIGS. 5 and 6, makespossible—in addition to a [size] adaptation in such a manner that thediffraction distribution of the diffraction-limited image 17 of the spot14 optimally fills out the input surface of the detector device 19—afurther operating mode, particularly if more than one Airy disk isimaged in the detection plane 18. In a measurement in which more thanone Airy disk is imaged on the detector device 19, light from furtherdepth planes of the sample 2 can be detected on the pixels of thedetector device 19 which are further outward. During the processing ofthe image, additional signal strengths are obtained without negativelyinfluencing the depth resolution of the LSM 1. The zoom lens 27 and/or29 therefore makes it possible to choose a compromise between thesignal-to-noise ratio of the image and the depth resolution.

The prerequisite for achieving the increase in resolution using thenamed methods is the fine scanning of the fluorescence light fielddistribution in the pinhole plane (detection plane). In order to keepthe data transfer- and data processing rate low, the configuration usesthe least possible number of detector (fiber) elements. This technicalapproach, in combination with the dependence of the dimensions of thefield distribution in the detection plane on the diameter of the lenspupil and the wavelength of the fluorescence, makes it necessary to beable to match the lens-specific dimensions of the sub-Airy fielddistribution in relative size to the fiber bundle, for optimum scanning.

The classic, widely-used approach of being able to control the size ofthe field distribution in the pinhole plane consists of using a zoomsystem in a pupil plane. As an alternative, a focusing lens with avariable aperture and fixed focus length can be used (see FIG. 11 aswell). This variant consists, inter alia, of fewer lenses than arerequired in a pupil/zoom system. This means that both the complexity andthe costs are lower in the case of the focusing lens. Only one fiberbundle is required, and the size of the fluorescence light is keptconstant.

If one would like to optionally, or completely, dispense with a zoomsystem or an adjustable focusing lens, and nevertheless ensure therequired lens- and/or wavelength flexibility, it is suggested accordingto the invention that multiple fiber bundles with different diameters bearranged next to each other, and the individual fibers of the bundles bemade to each preferably end at the same detector element of a detectorarray (FIG. 9, 10). The fiber bundle with the most suitable sampling ismoved onto the optical axis, or the primary color splitter in theillumination beam path is tilted in such a suitable manner (similarly toan adjustment of the pinhole by means of the primary color splitter)that the selected fiber bundle is illuminated, and only this light isdetected and ultimately included in the final accounting.

These methods of selecting the most suitable fiber bundle can beswitched very quickly (compared to a size modification of the Airy diskon a fiber bundle, by means of a zoom system), such that this solutionis particularly advantageous in the case of multi-spectral measurements.

Once the optimum adjustment of the focusing lens (FIG. 11) or the bestfiber bundle (FIG. 10) is found, the image contrast is increasedfollowing re-sorting of the data according to Sheppard et al. [2](particularly for higher spatial frequencies due to the increase inresolution achieved and/or to an improvement in the SNR). Thisoptimization of the scanning which is most suitable for the improvementof the resolution can be carried out in a closed regulating loop (aclosed loop) (FIG. 11: As a measure of finding the optimum adjustmentand/or the optimum fiber bundle, a combination of contrast andsignal-to-noise ratio is particularly suitable, because the optimumfocus is clearly defined by both conditions).

In the case of thick samples having a high fraction of light outside ofthe focus, the control parameters SNR favor a focusing lens adjustmentor a fiber bundle wherein multiple Airy disks are imaged on the fiberbundle. On the other hand, the contrast in a reconstructed image (areconstruction, by way of example, according to Sheppard et al., InOptik 80, No. 2, 53 (1982)) favors a detection light scanning whichpreferably only images one Airy disk on the fiber bundle, for optimumresolution. By means of a control variable, the system can be optimizedbetween resolution and SNR, wherein the properties of the sample usedare taken into account in the regulation.

For a regulation, the data at the start of the capture of an image canbe used, by way of example, to optimize the parameters (focusing lensadjustment within an image) (if the image already has representativeinformation at the edge thereof). As an alternative, the relevantinformation is first captured by means of snapshots, for the purpose ofoptimization for a selected region in the image, with reduced laserillumination and/or increased scanning speed.

An alternative to the opto-mechanical embodiments named above, for theoptimum (sub-) Airy scanning of the detection light, can be the use of asignificantly greater number of optical fiber and detector elements fromthe beginning, for example 2×2×32=128, necessary in principle. Theoptics must then not be variable.

In this variant, there are high data rates which cannot currently beprocessed by real-time computers. For this reason, it is advantageous todrop the data down from, by way of example the 128 channels to 32channels. For the case where precisely 1 Airy falls on the fiber bundle,4 channels, by way of example, are each combined in electronics whichare near to the detector (FPGA or a microcontroller), such that thecombined channels have the known arrangement. For the case in whichexactly 2 Airys are imaged by the fiber bundle, only the inner 32channels are read out. For intermediate values, a matching configurationmust be found depending on the size of the Airy disk, said configurationcombining fibers in a channel in order to achieve an increase inresolution in each lateral direction. An interpolation in the FPGA to,for example, 32 channels is likewise possible.

The invention is described below in greater detail with reference to theschematic illustrations in FIGS. 9-12.

The individual reference numbers (in addition to the reference numbersof the illustrations above) indicate:

-   -   40, 41, 42: fiber bundle    -   43, 44, 45: optical fiber    -   46: multi-channel detector    -   47: pivotable primary color splitter    -   48: slider    -   R: regulating loop    -   49: control unit/computer    -   50: adjustable focusing optics    -   51: signal assignment    -   52: signal assignment    -   53: fiber inputs    -   54, 55, 56: distribution of Airy diameter to the fiber inputs

FIG. 9 shows a schematic illustration of a laser scanning microscope asdescribed above with reference to the illustrations in FIGS. 1-8, withsub-Airy resolution detection in the pinhole plane, and a multi-fiberbundle arrangement.

In this case, multiple fiber bundles are applied to one and the samemulti-channel detector, which can be a PMT or APD array—or a PMT or APRDrow.

The optical fibers 43, 44, 45 originating from fiber bundles 40, 41, 42in this case, advantageously lie on the individual detector elements oneover the other with respect to their individual fiber ends, such that adetection of the light from the fiber ends of each optically activefiber bundle can be realized without a problem.

As an alternative, only every pair of two fiber ends can also lie oneabove the other on the respective detector element, and/or adjacentsegments of the detector can be used for the fibers of different fiberbundles.

The individual fiber bundles 40, 41, 42 advantageously apply their lightto the detector elements one after another, as a result of one of thefiber bundles being brought into position at the light spot which isimaged by the sample light on the input surfaces of the fiber bundles,as described further above.

In this case, 40-42 can be fiber bundles with different fiber diametersand/or fiber spacings.

An adaptation can be made by changing the fiber bundles, for example inthe case of a lens change for the lens O and/or in the case of a changeof the illumination wavelength.

A slider 48 is illustrated in FIG. 10, which captures the fiber bundles40-42 and which can slide perpendicular to the optical axis in order tobring the fiber bundles one after another into the optical axis. It isalso possible that the slider 48 only slides the aperture opening to amore suitable fiber bundle, and the flexible primary color splitterdeflects the detection light onto this more suitable fiber bundle.

A primary color splitter 47 which is able to pivot toward the opticalaxis is illustrated as an alternative, as a way of applying light todifferent fiber bundles 40-42 using its variable inclination.

FIG. 11 shows the optimization of the fluorescence light scanningaccording to the invention in a closed regulating loop, with a feedbacksignal.

An advantageous regulating loop R is illustrated in FIG. 11, and is alsoexplained with reference to FIG. 12.

In FIG. 11, an adjustment—for example of an adjustable focusing lens50—and a change of the fiber bundle, as described above in FIG. 10, iscarried out via a control unit/computer 49, utilizing an evaluation ofimage properties such as the image contrast—for example following thefinding of the Sheppard sum (which already has an improvedresolution)—or the signal-to-noise ratio or the image resolution.

When the focusing lens 50 and the fiber bundle 40-42 are optimallyfocused, the image contrast is increased as a result of an increase inthe resolution and/or improvement of the signal-to-noise ratio,particularly at higher spatial frequencies.

Therefore, an optimization of the captured signal can be advantageouslycarried out via the individual adjustments and settings described above,based on the respective conditions.

A combination of contrast and signal-to-noise ratio as a controlvariable is particularly suitable in this case, because the optimumfocus is clearly defined by both conditions.

An optimum evaluation process for the detection light can advantageouslycontribute in this case, by providing a combined reading of multipledetection fibers 43-45.

FIG. 12: Oversampling of the detection light and optimum binning toreduce the data transmission rate without losing resolution. A) Examplewith 128 fibers and detector elements. B) 1 Airy is imaged on the 128fibers and then binned by a factor of four. C) 2 Airys are imaged on the128 fibers; only the 32 inner channels are read out. D) 1.5 Airys on thetotal fiber array.

For the case where precisely 1 Airy falls on the fiber bundle, 4channels, by way of example, are each combined in electronics which arenear to the detector (FPGA or a microcontroller), such that the combinedchannels have the known arrangement (FIG. 12B). For the case in whichexactly 2 Airys are imaged by the fiber bundle, only the inner 32channels are read out (FIG. 12C). For intermediate values, a matchingconfiguration must be found depending on the size of the Airy disk onthe fiber bundle, said configuration combining fibers into one channelin order to achieve an increase in resolution in each lateral direction.An interpolation in the FPGA to, for example, 32 channels is likewisepossible. FIG. 12D) shows one example for the named situation.

As an alternative to the redistribution of the measurement signals, asin FIG. 12D, where each fiber is functionally assigned to exactly onechannel, it is possible to distribute the D signals of one fiber tomultiple channels in a FPGD, for example by means of algorithms whichare known in the art for the scaling of images.

Fiber inputs 53 are illustrated by way of example in FIG. 12A, whereinsubstantially one detector element 1-128 on the detector array isfunctionally assigned to each of these fiber inputs 53.

A combination of the signals of multiple detector elements isillustrated in FIG. 12B using fiber inputs 54 which are combined into 32segments, functionally assigned to the individual fibers for 32generated detection signals. This enables a reduction of the detectionchannels for the illustrated Airy diameter of the light spot AR.

As a result, it is advantageously possible to reduce the datatransmission rate without transmission losses.

In FIG. 12C, the Airy diameter AR (see FIG. 11) is smaller by a factorof 2, and is therefore scanned by a smaller number of fiber input ends.

In this case as well, only the detected inner channels are read out—inthis case the inner 32 from FIG. 12A.

The AR [Airy disk] in FIG. 12D is smaller than the fiber bundle by afactor of 1.5.

In this case, differently sized individual segments SG are used toillustrate that a different binning of read detection signals can alsobe carried out, by combining different numbers of fibers being combinedinto differently sized segments SG.

The different combination of the detection channels of the associatedindividual detectors of the detector array 56 via the optical fibers, asillustrated in FIGS. 12B-D, can advantageously be carried out at thesame time as—or prior to or after—an adaptation of the Airy diameter ofthe light spot by an adjustment via a variable lens 50. And anoptimization process is carried out via a regulating loop, as describedin FIG. 11, using the captured signal or the generated sample image,based on the criteria (contrast, etc.) described above.

The combination of the detection channels also constitutes anadvantageous alternative to the adaptation by means of a variable lens50.

In this case, an exchange of the fiber bundles, as in FIGS. 9, 10, canadditionally take place, and the optimization of the signal and/or imagecapture can also be carried out for this exchange.

This is performed, by way of example, by comparing each of multipleimages of the sample, or of a test sample, captured under differentconditions.

While the invention has been illustrated and described in connectionwith currently preferred embodiments shown and described in detail, itis not intended to be limited to the details shown since variousmodifications and structural changes may be made without departing inany way from the spirit of the present invention. The embodiments werechosen and described in order to best explain the principles of theinvention and practical application to thereby enable a person skilledin the art to best utilize the invention and various embodiments withvarious modifications as are suited to the particular use contemplated.

What is claimed is:
 1. A microscope for high resolution scanningmicroscopy of a sample comprising: an illumination device forilluminating the sample, an imaging device for scanning at least onepoint or linear spot across said sample and of imaging said point orlinear spot into a diffraction-limited, static single image having asize, a detector device detecting said single image in a detection planefor various scan positions, an evaluation device for evaluating adiffraction structure of said single image for said scan positions,using data from said detector device, and generating an image of saidsample which has a resolution which is enhanced beyond a diffractionlimit, said detector device including a detector array which has pixelsand a size which is larger than said single image, and a closed-loopcircuit configured to perform an adjustment by adjusting a zoomlens/focusing lens to adapt the size of said single image to the size ofsaid detector array using a feedback signal, and interconnectingindividual pixels of said detector array using a feedback signal, saidadjustment including image contrast and image sharpness andsignal-to-noise ratio as a control variable.
 2. The microscope accordingto claim 1, further comprising multiple non-imaging redistributionelements arranged in front of said detector array between said detectorarray and said detection plane, which one at a time distribute radiationemanating from said single image in said detection plane onto saidpixels of said detector array in a non-imaging manner, said multipleredistribution elements being fiber bundles which differ in number ofthe fibers and arrangement of the fibers and fiber diameters and shapeof the fiber cross-section, at least two optical fibers from differentredistribution elements of said multiple redistribution elements endingat the same pixel of said detector device, and said redistributionelements being arranged in a manner allowing pivoting or sliding theminto an optical axis detecting sample light.
 3. The microscope accordingto claim 2, wherein said redistribution element comprises a bundle ofoptical fibers of multi-mode optical fibers, which has an input arrangedin said detection plane, and an output where the optical fibers end atsaid pixels of said detector array in a geometric arrangement whichdiffers from that of said input.
 4. The microscope according to claim 2,wherein said optical fibers run from said input to an output in such amanner that optical fibers which are adjacent said output are alsoadjacent said input, in order to minimize a radiationintensity-dependent crosstalk between adjacent pixels.
 5. The microscopeaccording to claim 2, wherein said redistribution element has a mirror,said minor having different mirror elements comprising a multi-facetedmirror, a Digital Micromirror Device (DMD), or an adaptive mirror, whichdeflects radiation from said detection plane onto said pixels of saiddetector array, wherein said pixels of said detector array have ageometric arrangement which differs from that of said mirror elements.6. The microscope according to claim 1, wherein said imaging device hasa zoom lens arranged in front of said detection plane in the imagingdirection matching the size of said single image to that of saiddetector device.
 7. The microscope according to claim 6, wherein saidillumination device and said imaging device share a scanning device suchthat said illumination device illuminates said sample with adiffraction-limited point- or linear spot which coincides with said spotimaged by said imaging device, wherein said zoom lens is arranged insuch a manner that it is also a component of the illumination device. 8.The microscope according to claim 1, wherein said detector array is adetector row.
 9. The microscope according to claim 1, furthercomprising: for at least a part of said pixels of said detector array,radiation from at least two redistribution elements is functionallyassigned to each individual pixel, said redistribution elements beingarranged in a manner allowing pivoting or sliding into an optical axisdetecting sample light, a number of the individual signals of thedetector array being reduced by interconnecting individual pixels insaid signal evaluation, said detector pixels being only read out inregions which are at least illuminated with sample light, selectivechannels being read out and are combined for further processing in anevaluation circuit which is connected downstream of the detector arrayby a Field Programmable Gate Array (FPGA) or a microcontroller.
 10. Themicroscope according to claim 1, wherein said detector row is anAvalanche Photodetector (APD) row.
 11. The microscope according to claim1, wherein said detector row is a photomultiplier tube (PMT) row.
 12. Amethod for high resolution scanning microscopy of a sample, comprising:illuminating a sample, guiding at least one point or linear spot oversaid sample in a scanning manner so that it is imaged into a singleimage having a size, detecting said single image for various differentscan positions such that a diffraction structure of said single image isdetected, evaluating, said diffraction structure of said single imagefor each scan position and generating an image of said sample which hasa resolution which is enhanced beyond the diffraction limit, a detectorarray being included which comprises pixels and which has a size that islarger than said single image, and adjusting a zoom lens/focusing lensto adapt said size of said single image to said size of said detectorarray using a feedback signal, and interconnecting individual pixels ofsaid detector array using a feedback signal, the adjustment includingimage contrast and image sharpness and signal-to-noise ratio as aregulation parameters control variable.
 13. The method according toclaim 12, further comprising selectively redistributing radiationemanating from said single image in said detection plane on said pixelsof said detector array in a non-imaging manner by pivoting or sliding aselected one of multiple redistribution elements, located in front ofsaid detector array between said detector array and said detectionplane, into said optical axis detecting sample light, and said multipleredistribution elements being fiber bundles which differ in numbers offibers and arrangement of said fibers and fiber diameters and shape ofthe fiber cross-section, of which at least two optical fibers fromdifferent redistribution elements of said multiple redistributionelements end at individual pixels.
 14. The method according to claim 13,wherein said radiation of said single image is redistributed by means ofa bundle multi-mode optical fibers, which has an input arranged in thedetection plane, and an output where the optical fibers end at saidpixels of said detector array in a geometric arrangement which differsfrom that of said input.
 15. The method according to claim 14, whereinsaid optical fibers run from said input to said output in such a mannerthat optical fibers which are adjacent said output are also adjacentsaid input, in order to minimize a radiation intensity-dependentcrosstalk between adjacent pixels.
 16. The method according to claim 14,wherein said bundle of optical fibers and said detector array arecalibrated, by each optical fiber individually receiving radiation, byinterference signals in pixels which are associated with optical fiberswhich are adjacent thereto at said output being detected, and by acalibration matrix being established, by means of which a radiationintensity-dependent crosstalk between adjacent pixels is corrected insubsequent microscopy of the sample.
 17. The method according to claim14, wherein said radiation of said single image is redistributed bymeans of a mirror, said mirror having differently inclined mirrorelements wherein radiation from said detection plane is directed by saidmirror onto pixels of said detector array, and wherein said pixels ofthe detector array have a geometric arrangement which differs from thatof said mirror elements.
 18. The method according to claim 12, whereinsaid detector array is a detector row.
 19. The method according to claim12, further comprising determining a direction of movement of scanningof said point or linear spot by signals of individual pixels of saiddetector array being evaluated by means of cross-correlation.
 20. Themethod according to claim 12, further comprising detecting changes insaid sample by means of determining and evaluating a chronologicalchange in said diffraction-limited single image for said point- orlinear spot which is stationary in said sample.
 21. The method accordingto claim 12, further comprising: functionally assigning, for at least apart of said pixels of said detector array, radiation from at least tworedistribution elements to each of said individual pixels, saidredistribution elements being fiber bundles which differ in number offibers and arrangement of said fibers and fiber diameters and shape ofthe fiber cross-section, arranging said redistribution elements in amanner allowing pivoting or sliding into said optical axis detectingsample light, reducing said number of said individual signals of saiddetector array reduced by interconnecting individual pixels in saidsignal evaluation, said detector pixels only being read out in regionswhich are at least illuminated with sample light, making an adjustmentin a closed-loop circuit by changing redistribution elements, andadjusting a zoom lens/focusing lens to adapt image sizes, andinterconnecting individual pixels of said detector array using afeedback signal, said adjustment including image contrast and imagesharpness and signal-to-noise ratio as a control variable, and whereinselective channels are read out and are combined for further processingin an evaluation circuit which is connected downstream of said detectorarray by a Field Programmable Gate Array (FPGA) or a microcontroller.22. The method according to claim 17, wherein said mirror is amultifacet mirror.
 23. The method according to claim 17, wherein saidmirror is a Digital Micromirror Device (DMD).
 24. The method accordingto claim 17, wherein said mirror is an adaptive mirror.
 25. The methodaccording to claim 18, wherein said detector row is an AvalanchePhotodetector (APD).
 26. The method according to claim 18, wherein saiddetector row is a photomultiplier tube (PMT) row.